Near-infrared light-controlled DNA nano-platform composite material, preparation method and application in high-purity extraction of exosomes

Abstract

The application provides a near-infrared light-controlled DNA nano-platform composite material, a preparation method and application in high-purity extraction of exosomes, and belongs to the technical field of exosome separation. The near-infrared light-controlled DNA nano-platform composite material is prepared by growing long rolling circle amplification products containing multiple exosome surface specific expression protein CD63 aptamers on the surface of a photo-thermal responsive nano material through a solution phase rolling circle amplification reaction. The DNA nano-platform composite material can reversibly capture and separate exosomes with high efficiency and high purity under near-infrared light without adding any exogenous reagent, and compared with a differential separation method most commonly used at present, high-purity exosomes can be obtained; and the method is simple, time-saving, does not need expensive instruments and equipment, and is easy to be miniaturized and portable.

Description

Technical Field

[0001] This invention belongs to the field of exosome separation technology, and in particular relates to a near-infrared light-controlled DNA nanoplatform composite material, its preparation method, and its application in high-purity exosome extraction. Background Technology

[0002] Stem cells are a type of pluripotent cell that can differentiate into many different types within the body and can divide and proliferate indefinitely. In recent years, clinical studies have demonstrated that stem cells have numerous medical applications. Theoretically, stem cells or their derivatives could be used to repair any tissue lost or damaged due to disease or injury.

[0003] Stem cell exosomes are extracellular vesicles secreted by stem cells. Increasing research shows that stem cell exosomes have similar biological properties to stem cells, but they are safer, more stable, and more efficient, with stronger signal molecule transport and regulation capabilities. This is mainly due to the small size of exosomes, about 30-100 nm, which is at the nanoscale and easily absorbed and utilized by the body. Furthermore, since exosomes are not cells, they are easy to preserve and are not susceptible to xenografting, providing new strategies and methods for tissue regeneration and repair in cell-free transplantation therapy.

[0004] Despite the numerous advantages of stem cell exosomes, achieving high-purity capture and separation remains a challenge. Currently, the most commonly used exosome separation technique is differential centrifugation, but this method requires significant centrifugal force (100,000 g or more), is expensive and time-consuming, and most importantly, cannot separate vesicle subpopulations of similar size and density. Secondly, other exosome extraction methods, such as differential centrifugation and polymer precipitation, mostly require the addition of exogenous reagents, making subsequent separation and purification difficult. Recently, capturing exosomes through biomacromolecules has become possible, for example, through immunoassay, but this is accompanied by the drawback of not being able to achieve reversible release of exosomes and low material utilization. Therefore, obtaining high-purity exosomes without adding any exogenous reagents, while maximizing the preservation of their activity, is crucial in this field. Summary of the Invention

[0005] This invention provides a near-infrared light-controlled DNA nanoplatform composite material, its preparation method, and its application in the high-purity extraction of exosomes. Using the obtained composite material, high-purity exosomes can be obtained under near-infrared light without adding any exogenous reagents, and their activity can be preserved to the greatest extent.

[0006] To achieve the above objectives, the present invention provides a near-infrared light-controlled DNA nanoplatform composite material, which is prepared by growing a long rolling circle amplification product containing multiple exosome surface-specific protein CD63 aptamers through a solution-phase rolling circle amplification reaction on the surface of a photothermal responsive nanomaterial.

[0007] DNA, with its high programmability, predictable thermodynamics, and biocompatibility, has become an excellent template for designing nanomechanical devices and nanomachines that can respond to specific cues, thus making dynamic, remote, and even reversible responses. Inspired by the fact that the long tentacles of marine organisms contain multiple adhesive domains that can effectively capture flowing food particles, the inventors have constructed a DNA nanoplatform composite material to achieve efficient and highly specific capture of mesenchymal stem cell exosomes.

[0008] Near-infrared light possesses high tissue penetration and minimal photodamage. Furthermore, compared to other methods, photons offer high spatial and temporal controllability, leading to its widespread application in tumor therapy, protein function regulation, and cell behavior modulation in recent years. Noble metal nanoassemblies, such as magnetic beads / colloidal gold, are effective photothermal agents due to their unique localized surface plasmon resonance (LSPR) properties. CD63 is a protein specifically and highly expressed on the exosome surface. Based on proximity-dependent surface hybridization, CD63 on the exosome surface is captured by multiple DNA aptamers. The secondary structure of DNA aptamers is highly sensitive to temperature changes. Therefore, DNA aptamers can be used to capture exosomes, with magnetic beads / colloidal gold serving as the photothermal culture medium. Utilizing the LSPR properties of magnetic beads / colloidal gold, near-infrared light irradiation releases exosomes trapped in the closed-loop structure of the aptamers, achieving reversible exosome capture and release.

[0009] Preferably, the photothermal responsive nanomaterial is prepared by fixing colloidal gold onto magnetic beads, wherein the colloidal gold is prepared by sodium citrate reduction.

[0010] Preferably, the colloidal gold has a particle size of 13nm±2nm, and the magnetic beads have a particle size of 300nm±2nm.

[0011] This invention provides a method for preparing a near-infrared light-controlled DNA nanoplatform composite material according to any of the above technical solutions, comprising the following steps:

[0012] By fixing colloidal gold onto magnetic beads, a magnetic bead / colloidal gold composite material is obtained, which is a photothermal responsive nanomaterial.

[0013] Thiol-labeled rolling circle amplification initiating strand DNA was added to a solution containing magnetic beads / colloidal gold composite material and allowed to react fully to obtain magnetic beads / colloidal gold modified with initiating strand DNA.

[0014] Subsequently, a DNA circular template and Phi 29 DNA polymerase were added, and a solution-phase rolling circle amplification reaction was carried out on the surface of the magnetic beads / colloidal gold modified with the initiator strand DNA to generate a long rolling circle amplification product containing multiple CD63 aptamers, thus obtaining a near-infrared light-controlled DNA nanoplatform composite material.

[0015] Preferably, the mass concentration ratio of the added initiating DNA to the magnetic beads / colloidal gold is (500-600):1, and the initiating DNA and magnetic beads / colloidal gold are reacted at 37°C for 12 hours.

[0016] As a preferred embodiment, the solution-phase rolling ring amplification reaction specifically comprises:

[0017] The DNA circular template, 3U T4 ligase, 0.6μL 10mM dNTP, and 3U Phi 29 DNA polymerase were mixed thoroughly and reacted for 2–3 hours to obtain a long rolling circle amplification product containing multiple CD63 aptamers.

[0018] This invention provides an application of the near-infrared light-controlled DNA nanoplatform composite material according to any of the above technical solutions in the reversible capture and release of high-purity stem cell exosomes, wherein the stem cell exosomes specifically express CD63 on their surface.

[0019] Preferably, the stem cell exosomes are mesenchymal stem cell exosomes; the purity of the mesenchymal stem cell exosomes is ≥90%.

[0020] As a preferred option, the following steps are included:

[0021] The DNA nanoplatform composite material was added to the supernatant of mesenchymal stem cell culture medium and incubated for 30-40 minutes. The supernatant was removed by magnetic separation to obtain the DNA nanoplatform composite material / exosomes. After washing, it was stored in 1 mL of PBS.

[0022] Take 0.5 mL of DNA nanoplatform composite / exosome solution and expose it to an energy density of 1.055 W / cm² at 808 nm. 2 Under near-infrared light, the solution is rapidly heated to 45-50°C and held at that temperature for 10-12 minutes.

[0023] The DNA nanoplatform composite material was then removed by magnetic separation, and the resulting supernatant was the isolated mesenchymal stem cell exosomes.

[0024] In the above scheme, the DNA nanoplatform composite material serves as a photothermal reagent. After magnetic separation, when near-infrared light at 808 nm is applied, the photothermal conversion efficiency of the magnetic beads / colloidal gold allows the secondary structure of the CD63 aptamer to unwind, thereby releasing high-purity exosomes. Compared to a single aptamer, experiments have demonstrated that the DNA nanoplatform with multiple CD63 aptamer structures can achieve more efficient exosome capture.

[0025] Preferably, the volume ratio of the added DNA nanoplatform composite material to the supernatant of the mesenchymal stem cell culture medium is approximately 1:(50-60).

[0026] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0027] 1. The DNA nanoplatform composite material provided by this invention is based on the near-infrared light irradiation to induce heat generation of magnetic beads / gold nanoparticles to destroy the secondary structure of CD63 aptamers, thereby realizing the release of exosomes. No exogenous reagents are added during the process. Compared with the most commonly used differential separation method at present, high-purity exosomes can be obtained.

[0028] 2. The DNA nanoplatform composite material provided by this invention can not only capture but also separate exosomes, with high material utilization and good reproducibility; furthermore, when capturing exosomes, the DNA sequence captured by this composite material contains only CD63 aptamers specifically expressed on the surface of exosomes, exhibiting high specificity.

[0029] 3. The DNA nanoplatform composite material provided by this invention has good stability in cell culture medium and can be used in complex environments; moreover, the method of capturing and separating mesenchymal stem cell exosomes based on this composite material is simple, time-saving, does not require expensive instruments and equipment, and is easy to miniaturize and port. Attached Figure Description

[0030] Figure 1 A schematic diagram illustrating the fabrication process of the biomimetic DNA nanoplatform provided in an embodiment of the present invention;

[0031] Figure 2 This is a schematic diagram illustrating the capture and optically controlled separation of ultrapure exosomes using a biomimetic DNA nanoplatform provided in an embodiment of the present invention.

[0032] Figure 3 This invention provides a solution-phase rolling circle amplification experiment to verify the accuracy of the results.

[0033] Figure 4 The particle size and potential characterization diagrams provided in the embodiments of the present invention are for the material synthesis process.

[0034] Figure 5 Electron microscopy characterization of isolated exosomes provided in embodiments of the present invention;

[0035] Figure 6 A schematic diagram of WB characterization of MB / AuNPs / RCA capture of exosomes provided in an embodiment of the present invention.

[0036] Figure 7 This is a schematic diagram illustrating the efficient exosome capture of MB / AuNPs / RCA by WB characterization provided in an embodiment of the present invention. Detailed Implementation

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Example 1: Preparation of Photothermal Response Nanomaterials

[0039] The photothermal responsive nanomaterial is mainly composed of 300nm magnetic beads and 13nm colloidal gold (AuNPs). The 300nm magnetic beads were purchased from Thermo Fisher, and the 13nm colloidal gold was prepared by sodium citrate reduction.

[0040] The preparation method of photothermal responsive nanomaterials is as follows:

[0041] Preparation before the experiment: Soak all glassware in aqua regia (HCl / HNO3 = 3:1) for 30 minutes, rinse them with water, and then dry them in an oven.

[0042] 1.1 Preparation of 13nm colloidal gold: 100mL of 0.01% chloroauric acid was added to a round-bottom flask and refluxed using a condenser, while heating and vigorous stirring were performed. During stirring, 3.5mL of 1% sodium citrate was added, and the color of the solution changed from pale yellow to colorless, and finally to wine red. After boiling for 10 minutes, the heat source was removed, and the colloidal mixture was stirred until the solution reached room temperature. Then, it was filtered through a 0.45μm filter membrane, and the particle size was characterized by transmission electron microscopy (TEM) to be 13nm±2nm. Subsequently, it was stored in a refrigerator at 4℃ for long-term storage.

[0043] 1.2 Preparation of photothermal responsive nanomaterials (magnetic bead / colloidal gold composite material): 40 μL of 10 mg / mL carboxyl-modified magnetic beads were dispersed in 1.5 mL of 2-(N-morpholine) ethanesulfonic acid buffer (MES, 10 mM, pH 6.0, 150 mM NaCl), and 8 mg of EDC and 1.2 mg of NHS were added to the solution. The mixture was incubated in a constant temperature shaking water bath at 37 °C for 15 min. The carboxyl-activated magnetic beads were separated by magnetic separation and dispersed in 400 μL of phosphate buffer (PBS, pH 7.4, 150 mM NaCl). Then, 400 μL of 1 mM cysteine ​​was added to the above PBS buffer solution, and the mixture was incubated at 37 °C for 24 h. The cysteine-functionalized magnetic beads were separated again by magnetic separation and washed three times with 400 μL of PBS buffer. Finally, the cleaned magnetic beads were dispersed in 1 mL of colloidal gold solution, and the mixture was allowed to react for 24 h. Finally, the obtained magnetic bead / colloidal gold composite material (denoted as magnetic bead / colloidal gold) was magnetically separated again, and the obtained composite material was dispersed in 1 mL of PBS buffer.

[0044] Example 2: Preparation of DNA Nanoplatform Composite Material

[0045] 2.1 DNA-modified magnetic beads / colloidal gold: Initiator strand DNA of sulfhydryl-labeled rolling circle amplification ( Figure 1 The primer, containing the rolling circle amplification initiating DNA sequence 5'-HS-TTT TTT TTT TTT TTT CACC TCG CTC CCG TGA CACTAA-3', was added to a solution containing magnetic beads / colloidal gold, with a DNA:magnetic bead / colloidal gold mass ratio of 500:1. The reaction was carried out at 37°C for 12 hours. Then, 3M sodium chloride solution was added in eight portions to achieve a final sodium chloride concentration of 0.3M, allowing the initiating DNA to bind more extensively to the magnetic bead / colloidal gold surface. Finally, the obtained DNA-modified magnetic beads / colloidal gold (denoted as magnetic bead / colloidal gold / primer) was magnetically separated, washed, and resuspended in PBS buffer for storage.

[0046] 2.2 Solution-phase rolling circle amplification experiment as follows Figure 3 As shown, a is the circular template for rolling circle amplification (RoBAM), b is the initiating strand DNA for RoBAM, c is a mixture of a and b, d is a mixture of a and b with added T4 ligase, and e is a mixture of a and b with added T4 ligase, dNTPs, and Phi 29 DNA polymerase. Strip e shows a large amount of RoBAM product generated. Solution-phase RoBAM experiments were performed on magnetic beads / colloidal gold / primer surfaces (e.g.,...). Figure 1As shown): Magnetic beads / colloidal gold were reacted with a DNA circular template (Sangon Biotech, the sequence of which is 5'-P'-GAG CGA GGT GGG GTG AAA AAA AAA AAA AAAAAA AAA AAA TA GCA TTA GTG TCA CGG-3'), T4 ligase (3U), dNTPs (0.6μL, 10mM), and Phi29 DNA polymerase (3U) for 2 hours to obtain the final composite material magnetic beads / colloidal gold / RCA, i.e., the 3D DNA nanoplatform composite material. Afterwards, the mixture was magnetically separated, washed, and resuspended in PBS buffer for storage. The particle size and potential changes during the fabrication of the DNA nanoplatform composite material are shown below. Figure 4 As shown. Figure 4 As shown, the magnetic beads are gradually modified with gold nanoparticles, initiating DNA, and water and particle size gradually increase, and the potential also gradually becomes more negative.

[0047] Example 3: Application of near-infrared light-controlled DNA nanoplatform composite material in the reversible capture and release of ultrapure mesenchymal stem cell exosomes

[0048] 3.1 Mesenchymal stem cell culture: Mesenchymal stem cells were cultured in DMEM (10% fetal bovine serum, 1% penicillin and streptomycin, 1× non-essential amino acids, 10 ng / mL basic fibroblast growth factor and 10× mercaptoethanol) and in an incubator (Thermo Fisher) at 37°C and 5% CO2.

[0049] 3.2 Add 1 mg of the magnetic bead / colloidal gold / RCA composite material to 10 mL of mesenchymal stem cell culture medium supernatant and incubate for 30 minutes. Then, separate the components using magnetic separation, remove the supernatant, and obtain the magnetic bead / colloidal gold / RCA / exosomes. Add the separated magnetic bead / colloidal gold / RCA / exosomes to 1 mL of PBS, wash carefully 3 times, and then store in 1 mL of PBS.

[0050] 3.3 Take 0.5 mL of magnetic bead / colloidal gold / RCA / exosome solution and expose it to 808 nm at an energy density of 1.055 W / cm². 2 Under near-infrared light (focused photoelectric 808nm infrared laser), the solution was rapidly heated to 50°C and maintained at that temperature for 10 minutes. Then, magnetic beads / colloidal gold / RCA were removed by magnetic separation, and the resulting supernatant was the isolated ultrapure mesenchymal stem cell exosomes. The specific process is as follows... Figure 2 As shown.

[0051] Example 4: Characterization and Validation of Exosomes Isolated

[0052] Exosomes isolated by the DNA nanoplatform were placed in a copper grid and characterized using transmission electron microscopy, such as... Figure 5 As shown, the isolated exosome membrane structure remained intact, indicating that the exosomes extracted using this method were not damaged. The presence of CD63 was verified using Western blotting (WB), characterizing the successful capture and separation of exosomes. Exosome lysates were separated by 8% SDS-PAGE electrophoresis and then transferred to a nitrocellulose membrane for 12 minutes using a semi-dry electrophoresis transfer device. After blocking with 5% BSA-PBST (1×PBS and 0.1% Tween-20) solution for 1 hour, the membrane was reacted overnight at 4°C with a primary CD63 antibody (1:1000 dilution) and then reacted with a secondary CD63 antibody (1:5000 dilution) at room temperature for 1 hour. Before imaging, the membrane was reacted with ECL substrate solution (NCM Biotech Co., Ltd.). Chemiluminescence images were obtained using a chemiluminescence imaging system (ChemiDoc™). Results are shown below. Figure 6 As shown, magnetic beads alone, magnetic beads / gold nanoparticles, and magnetic beads / gold nanoparticles / primer do not capture exosomes. Exosomes are only captured after rolling circle amplification is induced on the surface of magnetic beads / gold nanoparticles to generate CD63 aptamers.

[0053] Example 5: Comparison with methods for capturing exosomes using a single aptamer and differential centrifugation

[0054] To demonstrate the advantage of generating multiple aptamers through rolling circle amplification on the surface of magnetic beads / gold nanoparticles, we compared our method with ligating a single CD63 DNA aptamer sequence onto the surface of only the magnetic beads / gold nanoparticles. We also compared this method with traditional differential centrifugation for exosome extraction, verifying the nanoplatform's ability to efficiently capture exosomes. Equal volumes of stem cell culture supernatant were compared using magnetic beads / colloidal gold / RCA, magnetic beads / colloidal gold single aptamer, and differential centrifugation methods. Figure 7 As shown, all three methods can achieve exosome capture. Using ImageJ to calculate WB grayscale values, the amount of exosomes captured by multiple aptamers (magnetic beads / colloidal gold / RCA) is 5 times that of a single aptamer, and 1.4 times that of differential centrifugation. Furthermore, the separation time using magnetic beads / colloidal gold / RCA is shorter, approximately 2-3 hours, while differential centrifugation takes over 12 hours.

Claims

1. A near-infrared light-controlled DNA nanoplatform composite material, characterized in that, It is prepared by the following method: By fixing colloidal gold onto magnetic beads, a magnetic bead / colloidal gold composite material is obtained, which is a photothermal responsive nanomaterial. Thiol-labeled rolling circle amplification initiating strand DNA was added to a solution containing magnetic beads / colloidal gold composite material and allowed to react fully to obtain magnetic beads / colloidal gold modified with initiating strand DNA. Subsequently, a DNA circular template and Phi 29 DNA polymerase were added, and a solution-phase rolling circle amplification reaction was carried out on the surface of magnetic beads / colloidal gold modified with priming strand DNA to generate a long rolling circle amplification product containing multiple CD63 aptamers, thus obtaining a near-infrared light-controlled DNA nanoplatform composite material. The sequence of the rolling circle amplification initiating strand DNA is 5'-HS-TTT TTT TTT TTT TTT C ACC TCG CTC CCGTGA CAC TAA -3'; The sequence of the DNA circular template is 5'-P'-GAG CGA GGT GGG GTG AAA AAA AAA AAA AAA AAA AAAAAA TA GCA TTA GTG TCA CGG-3'.

2. The near-infrared light-controlled DNA nanoplatform composite material according to claim 1, characterized in that, The photothermal responsive nanomaterial is prepared by fixing colloidal gold onto magnetic beads, wherein the colloidal gold is prepared by sodium citrate reduction.

3. The near-infrared light-controlled DNA nanoplatform composite material according to claim 1 or 2, characterized in that, The colloidal gold has a particle size of 13nm±2nm, and the magnetic beads have a particle size of 300nm±2nm.

4. The method for preparing the near-infrared light-controlled DNA nanoplatform composite material according to any one of claims 1-3, characterized in that, Includes the following steps: By fixing colloidal gold onto magnetic beads, a magnetic bead / colloidal gold composite material is obtained, which is a photothermal responsive nanomaterial. Thiol-labeled rolling circle amplification initiating strand DNA was added to a solution containing magnetic beads / colloidal gold composite material and allowed to react fully to obtain magnetic beads / colloidal gold modified with initiating strand DNA. Subsequently, a DNA circular template and Phi 29 DNA polymerase were added, and a solution-phase rolling circle amplification reaction was carried out on the surface of magnetic beads / colloidal gold modified with priming strand DNA to generate a long rolling circle amplification product containing multiple CD63 aptamers, thus obtaining a near-infrared light-controlled DNA nanoplatform composite material. The sequence of the rolling circle amplification initiating strand DNA is 5'-HS-TTT TTT TTT TTT TTT C ACC TCG CTC CCGTGA CAC TAA -3'; The sequence of the DNA circular template is 5'-P'-GAG CGA GGT GGG GTG AAA AAA AAA AAA AAA AAA AAAAAA TA GCA TTA GTG TCA CGG-3'.

5. The preparation method according to claim 4, characterized in that, The mass concentration ratio of the added initiating DNA to the magnetic beads / colloidal gold was (500-600):1, and the initiating DNA and magnetic beads / colloidal gold were reacted at 37°C for 10-12 hours.

6. The preparation method according to claim 4, characterized in that, The solution-phase rolling ring amplification reaction is specifically as follows: Mix the DNA circular template, 3 U T4 ligase, 0.6 μL 10 mM dNTP, and 3 U Phi 29 DNA polymerase thoroughly and react for 2-3 hours to obtain a long rolling circle amplification product containing multiple CD63 aptamers.

7. The application of the near-infrared light-controlled DNA nanoplatform composite material according to any one of claims 1-3 in the reversible capture and release of high-purity stem cell exosomes, characterized in that, The stem cell exosomes specifically express CD63 on their surface.

8. The application according to claim 7, characterized in that, The stem cell exosomes are mesenchymal stem cell exosomes; The purity of the mesenchymal stem cell exosomes is ≥90%.

9. The application according to claim 8, characterized in that, Includes the following steps: Add the DNA nanoplatform composite material to the supernatant of mesenchymal stem cell culture medium, incubate for 30-40 minutes, remove the supernatant using magnetic separation to obtain the DNA nanoplatform composite material / exosomes, wash and store in 1 mL PBS; Take 0.5 mL of DNA nanoplatform composite / exosome solution and expose it to an energy density of 1.055 W / cm² at 808 nm. 2 Under near-infrared light, the solution is rapidly heated to 45-50°C and held at that temperature for 10-12 minutes. The DNA nanoplatform composite material was then removed by magnetic separation, and the resulting supernatant was the isolated mesenchymal stem cell exosomes.

10. The application according to claim 9, characterized in that, The volume ratio of the added DNA nanoplatform composite material to the supernatant of the mesenchymal stem cell culture medium is 1:(50-60).

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